A method for the identification of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Two-dimensional electrophoresis on cellulose acetate membrane permits the clean separation of sulfated glycopeptide in a mixture of acidic glycans (glycosaminoglycans and acidic glycopeptides). Two systems were used. In system 1, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M barium acetate (pH 8.0) in the second dimension. In system 2, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M HCl in the second dimension. All of the acidic glycans on electrophoretogram were stained with alcian blue in 70% ethanol. On the other hand, sulfated glycans alone were made visible with alcian blue in 0.1 M HCl. Alcian blue in 70% ethanol or 0.1 M HCl, when combined with periodic acid-Schiff's reagent identified sulfated glycopeptides on cellulose acetate membrane.     

Glycosaminoglycans and acidic glycoproteins in rabbit uterus under estrogenic conditions.

Uterine slices obtained from the estrogen-treated rabbits were digested with pronase. Glycosaminoglycans and acidic glycopeptides were then isolated by Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane (Separax), in succession. Each subfraction thus obtained was identified by the mobility on Separax electrophoresis and the digestibility with mucopolysaccharidases (Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase AC, chondroitinase ABC and heparinase). The resulting data showed that each complex saccharide (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide and sialoglycopeptide) was separated into 2-5 fractions, indicating charge and/or molecular heterogeneity of each complex saccharide.     

Metabolic rate of acidic complex saccharides in rabbit uterus under estrogenic condition.

Uterine slices obtained from estrogen-treated rabbits were incubated in vitro with N-acetyl-D-[1-3H]glucosamine together with D-[U-14C]glucose. The isotope-labelled acidic complex saccharides were then isolated by pronase digestion, Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane, in succession. In this way, individual acidic complex saccharides (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide, and sialoglycopeptide) were separated into 2-5 subfractions. The specific radioactivity of hexosamine in the subfractions indicated that the metabolic rate of the uterine complex saccharides as follows: hyaluronic acid greater than sulfated glycopeptide greater than heparan sulfate greater than chondroitin sulfate C greater than dermatan sulfate. In addition, metabolic heterogeneity of heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate was suggested.     

Isolation,Characterization and Synthesis of Pimprinine,Pimprinethine and Pimprinaphine,Metabolites of Streptoverticillium olivoreticuli

The sulfated glycopeptides in ovomucin, chalazae and yolk membrane were isolated from the proteolytic digests by gel filtration on a Bio-Gel P-100 column and DEAE-Sephadex A-25 column chromatography. These sulfated glycopeptides contained N-acetylhexosamine (23.3-26.8%), hexose (23.6-24.4%), sialic acid (11.2-18.0%), sulfate (5-12.1%) and peptide (17.5-18.1%). The sulfate contents of glycopeptides in chalazae and yolk membrane were much higher than those in ovomucin, about two times in a molar ratio to hexosamine. The sedimentation patterns of each sulfated glycopeptide were single and the sedimentation constants were around 3 S, suggesting that these sulfated glycopeptides were macromolecular components. Thus, the presence of highly sulfated glycoproteins was confirmed in chalazae and yolk membrane, which were different from those in ovomucin.     

Glycosaminoglycans and acidic glycoproteins in rabbit uterus under estrogenic conditions

Uterine slices obtained from the estrogen-treated rabbits were digested with pronase. Glycosaminoglycans and acidic glycopeptides were then isolated by Dowex 1 column chromatography and preparative electrophoresis on celulose acetate membrane (Separax), in succession.Each subfraction thus obtained was identified by the mobility on Separax electrophoresis and the digestibility with mucopolysaccharidases (Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase AC, chondroitinase ABC and heparinase). The resulting data showed that each complex saccharide (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide and sialoglycopeptide) was separated into 2–5 fractions, indicating charge and/or molecular heterogeneity of each complex saccharide.     

Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag

Glycan microarray technology has become a successful tool for studying protein–carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by Pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core α1,3-fucose and core α1,2-xylose. After simple Fmoc deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.     

Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.     

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.     

Altered glycoprotein synthesis in mouse epidermal cells treated with retinyl acetate in vitro

Retinyl acetate alters glycoprotein synthesis in mouse epidermal cells in culture. Epidermal glycoproteins were enzymatically digested to glycopeptides and separated on DEAE Sephadex A50 columns using different concentrations of LiCl. There was a two-fold increase in incorporation of fucose and glucosamine in the 0.2 M LiCl fraction from cells treated for 3 weeks with 12.5 μg/ml retinyl acetate and 1.25% DMSO as compared with DMSO controls. No changes were noted in other fractions. The glycopeptide from A treated cells isolated on 0.2 M LiCl had a higher molecular weight than glycopeptides from that same fraction eluted by control cells. This isolated newly synthesized glycopeptide from vitamin A treated cells appears to be a single product by rechromatography on DEAE Sephadex A50 and Sephadex G100 columns.     

A simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano LC coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry

In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method. Upon isolation, glycopeptides may be subjected to further glycoproteomic and glycomic workflows for the purpose of identifying glycoproteins present in the sample and characterizing their glycosylation sites, as well as their global and site-specific glycosylation profiles at the glycopeptide level. Detailed structural analysis of glycoforms may then be performed at the glycan level upon chemical or enzymatic release of the oligosaccharides. Aiming at complementing other purification methods, this technique is extremely simple, cost-effective, and efficient. Glycopeptide enrichment was verified and validated by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining electron-transfer dissociation (ETD) and collision-activated dissociation (CAD) fragmentation techniques.     

Cell surface glycoproteins I: Accumulation of a glycoprotein on the outer surface of mouse LS cells during mitosis

Surface glycopeptides derived from vertebrate cells have been separated into 4 classes by chromatography on DEAE cellulose columns. Among different cell types tested, significant differences were observed in the relative amounts of these 4 glycopeptide classes present on the cell surface. This type of heterogeneity is consistent with the expected biological role of cell surface glycoproteins. One glycopeptide, as revealed by the DEAE column analysis, was found to have a characteristic metabolic pattern in mouse LS cells. New accumulation of this structure, called glycopeptide 4, on the cell surface was detected only around the period of cell division (M phase) and not at other times during the cell cycle.     

Developmental Change in the Amount of Polysialosyl Glycopeptides Isolated from the Rat Brain

Polysialosyl glycopeptides were coisolated with glycosaminoglycans by Pronase digestion of the whole brains of perinatal rats and could be separated from known glycosaminoglycans by two-dimensional electrophoresis on cellulose acetate film. The polysialosyl glycopeptides could not be obtained from fetal rat brain on day 13 of gestation, but began to be detected on day 14. The amount of polysialosyl glycopeptides was estimated from the dye concentration of the Alcian blue-stained spot in the electrophoretogram. The glycopeptide content increased almost linearly, on the basis of brain DNA, up to 10 days after birth. Thereafter, the content decreased rapidly, and hardly any polysialosyl glycopeptides could be isolated from the brain at approximately 30 days. This developmental change may be involved in morphogenesis and maturation of the brain. The polysialosyl glycopeptides could be isolated from the cerebellum, from the cerebrum, or from the brainstem of the neonatal rat. However, each region of the brain had a postnatal developmental change in glycopeptide content different from those of the other regions.     

Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites

Proteomic techniques, such as HPLC coupled to tandem mass spectrometry (LC-MS/MS), have proved useful for the identification of specific glycosylation sites on glycoproteins (glycoproteomics). Glycosylation sites on glycopeptides produced by trypsinization of complex glycoprotein mixtures, however, are particularly difficult to identify both because a repertoire of glycans may be expressed at a particular glycosylation site, and because glycopeptides are usually present in relatively low abundance (2% to 5%) in peptide mixtures compared to nonglycosylated peptides. Previously reported methods to facilitate glycopeptide identification require either several pre-enrichment steps, involve complex derivatization procedures, or are restricted to a subset of all the glycan structures that are present in a glycoprotein mixture. Because the N-linked glycans expressed on tryptic glycopeptides contribute substantially to their mass, we demonstrate that size exclusion chromatography (SEC) provided a significant enrichment of N-linked glycopeptides relative to nonglycosylated peptides. The glycosylated peptides were then identified by LC-MS/MS after treatment with PNGase-F by the monoisotopic mass increase of 0.984 Da caused by the deglycosylation of the peptide. Analyses performed on human serum showed that this SEC glycopeptide isolation procedure results in at least a 3-fold increase in the total number of glycopeptides identified by LC-MS/MS, demonstrating that this simple, nonselective, rapid method is an effective tool to facilitate the identification of peptides with N-linked glycosylation sites.     

Convenient synthesis of a sialylglycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide

Access to glycopeptides with C-terminal thioester functionality is essential for the synthesis of large glycopeptides and glycoproteins through the use of native chemical ligation. Toward that end, we have developed a concise method for the synthesis of a glycopeptide thioester having an intact complex-type dibranched disialyl-oligosaccharide. The synthesis employed a coupling reaction between benzylthiol and a free carboxylic acid at the C-terminus of a glycopeptide in which the peptide side chains are protected. After construction of glycopeptide on the HMPB-PEGA resin through the Fmoc-strategy, the protected glycopeptide was released upon treatment with acetic acid/trifluoroethanol and then the C-terminal carboxylic acid was coupled with benzylthiol at -20 degrees C in DMF. For this coupling, PyBOP/DIPEA was found to be the best for the formation of the thioester, while avoiding racemization. Finally, the protecting groups were removed in good yield with 95% TFA, thus affording a glycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide.     

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain: Characterization after fractionation by concanavalin A-Sepharose affinity chromatography

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.     

Glycopeptide microarray for autoantibody detection in cancer

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860-1871 (2011). Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.     

Purification and characterization of two glycopeptide hydrolases from jack beans

Two glycopeptide hydrolases, an endo-beta-N-acetylglucosaminidase and peptide:N-glycanase (amidase), have been isolated from defatted jack bean meal by standard procedures involving differential solubility and column chromatography. The purified products appear to be free of contaminating proteases and exoglycosidases, and their substrate specificity has been explored with regard to both glycan and peptide structure of the substrates. The endoglycosidase appears to be specific for high mannose glycans; no hydrolysis of either hybrid or complex glycans has been observed. It shows limited activity with two intact glycoproteins, ribonuclease B and yeast invertase, and gives optimal rate with glycopeptides. Free glycan-Asn derivatives are poor substrates in comparison with glycopeptides or glycan-Asn derivatives where the alpha-amino group has been dansylated. The amidase will liberate both high mannose, hybrid, and asialo-complex glycans from both proteins and peptides, but many glycans in intact proteins or in long peptides are resistant to the amidase and become active as substrates only after further proteolytic cleavage. The best substrates appear to be those with the glycosylated asparagine no more than 4-5 residues in from either the NH2- or COOH-terminal end of the peptide. Sialylated glycans do not appear to be released by the amidase.     

Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells

From cells of a nullipotential line of embryonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% of the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an approximately 10-fold purification of the labeled material. Extreme heterogeneity of membrane glycoproteins labeled with these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 column were the predominant glycopeptide species, while complex glycopeptides of molecular weight around 2500 were minor components. Therefore, these large glycopeptides, characteristic of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slightly with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experiments excluded the possibility that the larg glycopeptides are mucopolysaccharides, glycolipids or mucin-type glycoproteins with short oligosaccharide chains.